Lack of knowledge of the structure of its major transactivation domain (AF1) has hampered full understanding of the mechanism by which the glucocorticoid receptor functions. It is presumed that AF1 interacts directly, or through intermediary proteins, with the "transcription machinery" complex of proteins. This requires surface-to-surface interactions between proteins and the structure to produce them. When expressed separately as a recombinant protein, however, AR displays little or no structure and binds weakly at best with a few such proteins. We have made two observations that offer exciting promise for understanding how AF1 obtains the structure necessary for its presumed function and for discovering its natural binding partners. First, we found that in the presence of an osmolyte, trimethylamine oxide (TMAO), the AF1 of the human GR acquires native-like structure and shows greatly enhanced binding to certain other proteins. We will use TMAO and other osmolytes to study the structure of AF1 and to find its binding partners. Second, we have shown that when a two-domain fragment of the GR containing AF1 and the DNA-binding domain is bound to its cognate DNA binding site, AR seems to acquire structure and be able to bind certain other proteins. In this project, we will determine the protein and DNA parameters that control this event. We will compare the AF1 structure and the protein binding partners uncovered by the two techniques. One of the nuclear proteins shown to bind AF1 under the conditions we employ appears to be a member of the nuclear receptor co- activator family of proteins. Methods used will include circular dichroism, intrinsic fluorescence emission spectroscopy, peptide mapping, thermodynamically rigorous studies of protein:DNA binding NMR and if crystals are obtained, X-ray diffraction.